1. Field of the Invention
The invention relates, generally, to the field of detection of proteins involved in blood coagulation. More specifically, it relates to a method for determining the amount of tissue factor and/or factor VIIa in simple and complex biological systems.
2. Description of the Related Art
Tissue factor (TF) and factor VIIa (fVIIa) are essential components for the initiation of blood coagulation. Blood coagulation is initiated when cryptic TF becomes exposed on the surface of vascular cells where it can bind circulating fVIIa.
Although several assays for fVIIa have been described (some commercially available), those assays do not discriminate between factor VII and factor VIIa, either due to the lack of specificity in immunologic methods, or due to the feedback-activation of factor VII in the amidolytic and clot-based assays. For example, there is a “direct” factor VIIa assay based upon clotting of plasma initiated with a soluble mutant of TF. The assay involves the entire coagulation cascade, so it is therefore sensitive to the concentration of procoagulant proteins and coagulation inhibitors as well as factor VII by virtue of feed-back activation. Thus, the clotting time of plasma reflects the concentration of fVIIa, as well as, the concentration of all components of plasma involved in coagulation and its regulation.
Moreover, because of the critical role TF plays in hemostasis, its potential role in metastasis, and its extensive use in-vitro, it is important to have a sensitive and specific TF assay that can detect relatively low amounts of this protein in biological fluids, cell cultures, lysates, and in purified and semi-purified systems. TF assays thus far developed employ clotting, chromogenic, and immunochemical methods. The clotting methods involve the entire coagulation cascade and are therefore sensitive to alterations in the levels of procoagulant proteins and coagulation inhibitors. Chromogenic methods do not allow a direct measure of TF activity, and are expensive since they require additional purified coagulation factors. Similarly, immunochemical methods are relatively expensive and time-consuming. Thus, at the present time, there is no quick, accurate and somewhat universal method to directly measure TF activity.
Accordingly, a functional-based assay that could be used to measure TF or fVIIa in purified and/or complex biological systems would have a variety of potential applications. These include: in-vitro diagnostics for the assessment of hemostatic potential; in-vitro diagnostics for thrombotic risk assessment; in-vitro diagnostics for cancer screening; quality control during the purification of recombinant tissue factor; quality control during the manufacture of prothrombin time PT reagents; and characterization of final TF and/or PT reagents.
It has been demonstrated that the amidolytic activity of the TF/factor VIIa complex toward small fluorogenic substrates is membrane (phospholipid) independent. This suggests that TF can be successfully quantitated in a free form in purified systems and biological fluids, as well as, present on cell or artificial membranes and in cell lysates. Fluorogenic substrates, which allow a quantitation of low concentrations of factor VIIa (as described above), will similarly allow the quantitation of low concentrations of TF. U.S. Pat. No. 5,399,487, which is fully incorporated by reference, discloses fluorogenic substrates for serine proteases that contain 6-amino-1-naphthalenesulfonamide (ANSN) leaving groups.